1. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/17/2006Chapter 9Capillary Electrophoresis Chapter 9Capillary Electrophoresis (CE) References: Dale R. Baker, Capillary Electrophoresis, John Wiley & Sons, 1995. M.G. Khaledi, Ed., High-Performance Capillary Electrophoresis, John Wiley & Sons, 1998. Colin F. Poole, The Essence of Chromatography, Elsevier, 2003.

2. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis 3.Modes of CE 3.1 Capillary Zone Electrophoresis (CZE) CZE or Free solution capillary electrophoresis (FSCE).  Fill capillary with a buffer of constant composition  Fill source and destination vials with same buffer  Analyze cations and anions simultaneously  Nor for neutral species

3. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2008Chapter 9Capillary Electrophoresis

5. 3.2Micellar electrokinetic capillary chromatography (MEKC) (Electrochromatography)  Developed by Shigeru Terabe et al. (Anal. Chem. 1984, 56, 111)  Provides a method for separation of electrically neutral compounds  Combines the separation mechanism of chromatography with the electrophoretic and electroosmotic movement of solutes and solutions  Separation is based on the partitioning of solutes between the surfactant micelles and the run buffer.  The detector output is referred to as electrokinetic chromatogram

6. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis

7. Three factors affecting apparent electrophoretic mobility of an anlyte in MEKC:  The system µ EOF  The fraction of analyte in the electrolyte solution and its µ EP  The fraction of analyte in the pseudo-stationary phase and the µ EP of the micelle

8. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis 3.2.1Principles of MEKC This mode of CE is based on the partitioning of solutes between micelles and the run buffer.  Detergents (surfactants) Molecules that have a hydrophilic, water soluble moiety on one end of the molecule and a hydrophobic, water insoluble moiety on the other. e.g. Sodium dodecyl sulfate (SDS), [CH 3 -(CH 2 ) 11 -O- SO 3 - ], also called sodium lauryl sulfate

9. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis  Critical micelle concentration (CMC) The concentration of a detergent present in solution when micelles begin to form.  Micelles Aggregations of individual detergent molecules.  Aggregation number The number of detergent that make up the a micelle.  Kraft point The temperature at which the solubility of the detergent = the critical micelle concentration

10. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis

11. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2005Chapter 9Capillary Electrophoresis

12. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis

13. Differences between chromatography and MEKC:  Chromatographic separations are based differences in distribution of sample molecules between a stationary phase and a mobile phase. However, in MEKC, there are two phases, aqueous and micelle, both of which move.  In chromatography, the solutes and mobile phase are moved through the column by pumped flow, whereas in MEKC, they are moved through the capillary by EOF.

14. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis

16. Retention Parameters in MEKC Capacity factor (retention factor) of an electrically neutral solute is defined the ratio of the umber of moles of solute in the micelles, n mc, to the number of the moles in the aqueous phase, n aq : k’ = n mc /n aq [3-1] k’ = (t R – t 0 )/t 0 (1 – t R /t mc )[3-2] (Terabe et al. Anal. Chem. 1984, 56, 111) This is similar to the equation in Chromatography k’ = (t R – t 0 )/t 0 [3-3] Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis

17. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2008Chapter 9Capillary Electrophoresis k’ = (t R – t 0 )/t 0 (1 – t R /t mc )(3-2) k’ = (t R – t 0 )/t 0 (3-3) (1 – t R /t mc ) is due to the retention properties of MEKC. When T mc is very large, eq (3-2) is same as (3-3). k’ is related to T 0, T R, and T mc in MEKC

18. Retention Parameters in MEKC Because t R = l/ s, t 0 = l/ 0, t mc = l/ mc : k’ = (t R – t 0 )/t 0 (1 – t R /t mc )[3-2] Insert t R = l/ s, t 0 = l/ 0, t mc = l/ mc and rearrange k’ = ( 0 / s – 1)/(1 - mc / s ) [3-4] Since =  E k’ = (  EOF /  s – 1)/(1-  mc /  s ) [3-5] Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2008Chapter 9Capillary Electrophoresis

19. Resolution in MEKC Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis Baker, 1995

20. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis 3.2.2Separation of ionic solutes by MEKC  Neutral molecules: Differences in their distribution between the aqueous buffer and the micelles  Ionic molecules: Differences in their electrophoretic mobilities or because of interactions with micelles, depending on the charges of the ionic solutes and the micelles.

21. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis For negatively charged micelles are used, such as SDS: Baker, 1995 cations neutral

22. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis 3.2.3Using Modifiers in MEKC  Modifiers affecting the charge on the micelle or the solute and changing the solubility of a solute in the micelles. e.g. addition of tetraalkylammonium (TAA) to an SDS buffer to improve the separation of carboxylic acids (formation of neutral ion pairs ). Affects also the retention of positively charged solutes (decreases when TAA salts were added).

23. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis  Modifiers serving as a second pseudophase Cyclodextrin-modified MEKC has been used to separate very hydrophobic solutes. Cyclodextrins (CD’s) are water-soluble oligosacchrides. CD’s have a characteristic toroidal shape, with a hydrophobic cavity and a hydrophilic external surface. CD’s are electrically neutral, they migrate with the velocity of the EOF Hydrophobic solute is not separated if no CD is added. When a CD is added to the buffer,

24. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis k’ = n mc /n CD

25. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis 3.3CAPILLARY ISOELECTRIC FOCUSING (CIEF) 3.3.1Properties of Amphiprotic Compounds An amphiprotic compound is a species that in solution is capable of both donating and accepting a proton. A typical amino acid, such as glycine, is an amphiprotic compound. CIEF is used to separated amphiprotic species, such as amino acids and proteins that contains a weak carboxylic acid group and a weak base amine group.

26. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis When glycine is dissolved in water, three important equilibrium: NH 2 CH 2 COOH  NH 3 + CH 2 COO - [1] Internal acid/base reaction proceeds far to the right, with product being the predominant species in the solution. NH 3 + CH 2 COO - + H 2 O  NH 2 CH 2 COO - + H 3 O + [2] K a = 2 x 10 -10 NH 3 + CH 2 COO - + H 2 O  NH 3 + CH 2 COOH + OH - [3] K b = 2 x 10 -12

27. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis zwitterion: The amino acid product bearing both a positive and a negative charge, is called a zwitterion. Isoelectric point (PI): The isoelectric point of an amphiprotic compounds is the pH at which the compound has a net charge of zero. No net migration of amino acid occurs in an electric field when the pH of the solvent is such that the concentrations of anionic and cationic forms are identical.

28. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis The PI is readily related to the ionization concentrations for the species. For glycine: K a = [H 3 O + ][NH 2 CH 2 COO - ]/[NH 3 + CH 2 COO - ] K b = [OH - ][NH 3 + CH 2 COOH]/[NH 3 + CH 2 COO - ] At isoelectric point, [NH 2 CH 2 COO - ] = [NH 3 + CH 2 COOH] Thus, K a /K b = [H 3 O + ]/[OH - ] Substitution of K w /[H 3 O + ]for [OH - ], then [H 3 O + ] = (K a K w /K b ) 2 [H 3 O + ] = 1 x 10 -6 pI = 6

29. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis Three Steps involved in CIEF:  Formation of a pH gradient in the capillary  Performing Isoelectric Focusing  Mobilization of the focused zones

30. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis 3.3.2Formation of a pH gradient in the capillary In isoelectric separation of amphiprotic species, the separation is performed in a buffer mixture that varies in pH continuously along its length. The pH gradient is prepared from the mixture of several different ampholytes in an aqueous solution..

31. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis NaOH H 3 PO 4

32. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis To form a pH gradient in a capillary: (1) The capillary is filled with a mixture of ampholytes that will produce a certain pH gradient (e.g. 3-10). (2) One end of the capillary is then inserted in a solution of strong base (NaOH) (cathode). The other end is immersed in a solution of strong acid (phosphoric) (anode). (3) When an electric field is applied, hydrogen ions begin to migrate from the anode toward the cathode, while hydroxide ions begin to move in the opposite direction. (4) The ampholytes in the buffer mixture migrate also depending on their net charge. Ultimately they reach the pH where their net charge is zero (pI).

33. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis 3.3.3Isoelectric Focusing  CIEF is performed by filling the capillary with a mixture of ampholytes and SAMPLE.  Similar to the migration of ampholytes, analyte ions also migrate until they reach their pI.  No EOF in the capillary. Large volumes of sample are injected.

34. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis 3.3.4Mobilization of the focused zones Hydrodynamic flow: to pressure the capillary. Electrophoretic mobilization Add NaCl into the NaOH solution after focusing. Both Cl - and OH - migrate into that end of the column, and sum of these two concentrations is balanced by H +. The pH gradient is no longer stable Analytes change to positively charged, and moves toward the cathode.

35. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/27/2006Chapter 9Capillary Electrophoresis

37. Dr. Bruce McCord November 6, 2006 3.4 Capillary Gel Electrophoresis